Extraction and High Performance Liquid Chromatographic Enrichment of Polyphenol Oxidase from Theobroma cacao Seeds

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Abstract
Polyphenol oxidase (PPO) was extracted from cocoa beans and subsequently purified using ammonium sulfate precipitation followed by high performance liquid chromatography (HPLC) for enrichment. HPLC analysis was performed using hydrophobic interaction chromatography which resulted in a 6.5‐fold enrichment and a 24.6% recovery of the enzyme. Enrichment was confirmed using polyacrylamide gel electrophoresis under nondenaturing conditions. Comparison of enzyme‐ and protein‐stained electrophoretic gels revealed the presence of possible multiple molecular forms of PPO and the absence of non‐enzymic contaminating protein, indicating a rapid procedure of enzyme extraction and enrichment.